PROJECT 6 PROJECT SUMMARY Alkylation therapy is used every day in the clinic but the absence of predictive mechanistic knowledge limits efforts to inform and advance its use. Project 6 seeks this foundational knowledge and targets ALKBH3 and ASCC alkylation repair proteins and complexes that have been linked to cancer malignancy. ALKBH3 is a direct damage reversal dealkylase that works on RNA and ssDNA, and ASCC is a multifunctional, heterotrimeric helicase (ASCC1, ASCC2, and ASCC3 subunits). It is not known why these two proteins are linked to cancer malignancy and what their cellular targets are. The central hypothesis for Project 6, based on recent findings by the Project 6 team, is that ALKBH3-ASCC-mediated alkylation repair of both DNA and RNA is critical for alkylation damage responses in some cancer cells. Project 6 will determine cellular targets of alkylating agents, how ASCC recruitment to alkylating agent-induced foci are coordinated with other DNA processes, and define informative and biologically-relevant structures and assemblies. To achieve this, Project 6 will take advantage of the resources available in the SBDR Program Project and will directly collaborate with Projects 1, 2, 3 and 5, plus with the SCB and EMB Cores, and provide thematic synergy with all Projects. The Project 6 team is led by Dr. Nima Mosammaparast (Washington University), who has made groundbreaking discoveries including the dependence of certain cancer cells on ALKBH3 and ASCC3, the first observation of alkylating agent-induced foci, and the determination that these foci are not associated with typical DNA break repair proteins but with elongating transcription complexes. Roopa Thapar (MD Anderson) will bring her RNA expertise and do NMR and biophysical analysis. Susan Tsutakawa (LBNL) will do Small Angle X-ray Scattering (SAXS) and crystallography. Yuan He, Project 1 collaborator, will do Cryo-Electron Microscopy (CryoEM). SBDR PI John Tainer will insure coordination with other Projects and Program goals. Overall Project 6 Aim 1 uses cell biology and biochemistry to identify functionally-relevant ASCC-ALKBH3 assemblies and to test hypotheses on DNA and RNA repair activities and their intersection with other repair pathways in cells. Aim 2 employs structural biology (NMR, crystallography, SAXS, and CryoEM) and biophysics to characterize active sites and interfaces that inform mechanisms and enable mutational validations. With our preliminary data, robust assays, and systems for producing and characterizing ASCC proteins and complexes in cells and in vitro, we are poised to add RNA and DNA alkylation repair to SBDR4. We will define biology- driven structures, mechanisms, and separation-of-function mutations to paradigm shift alkylation cancer research and provide powerful tools to examine alkylating therapies in cancer. Our results will directly improve SBDR?s ability to inform cancer researchers about RNA and DNA alkylation responses with impacts to cancer research, therapy and patient care decisions. The expected results will fill gaps in SBDR for a predictive mechanistic knowledge of DNA and RNA damage responses likely to impact cancer patient care.